Development of integrated biological processing for the biodesalination of sulphate- and metal-rich wastewaters
- Authors: Boshoff, Genevieve Ann
- Date: 1999
- Subjects: Sewage -- Purification -- Biological treatment Sulfates Mineral industries -- Environmental aspects
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3899 , http://hdl.handle.net/10962/d1003958
- Description: The substantial pollution threat to the South African environment from acid mine drainage (AMD) effluents has been well documented. Due to the juvenile nature of acidity in these flows, any remediation strategies implemented will need to function effectively and at low cost for long periods of time. The widespread use of sulphate reducing biological systems for the treatment of such effluents, and in particular large volume flows, has been limited. The supply of inexpensive electron donor and carbon sources, as well as appropriate reactor designs capable of handling large volume flows, have been identified as among the principal factors limiting development of this technology. The broad aim of the research programme reported here was to undertake an evaluation of the feasibility of an algal-bacterial integrated ponding system for the treatment of AMD, and the waste stabilisation pond (WSP) as an appropriate reactor design for this application. The study attempted to demonstrate the feasibility of individual unit operations in a proposed process train using complex organic carbon serving as the electron donor source for the sulphate reducing bacteria (SRB). Studies were undertaken as laboratory and pilot-scale investigations. Tannery effluent was shown to be a functional carbon source for biological sulphate reduction, with effective removal of sulphate and organics being recorded. In turn, the use of biological sulphate reduction for the treatment of tannery effluent was demonstrated. Algal biomass was shown in laboratory studies to function as an effective carbon source for biological sulphate reduction. It is known that micro-algae produce large quantities of photosynthate which is released to the growth medium under conditions of physiological stress. The potential for the use of photosynthate production in high rate algal ponding systems and its manipulation and use as a sustainable carbon source for sulphate reduction was investigated. Growth of a mixed culture of Dunaliella under conditions of light, temperature and salinity stress demonstrated production of large quantities of organic carbon. However, growth was inhibited at high temperatures. An elevation of salinity levels led to a decrease in growth of Dunaliella, but to increased organic carbon production. Spirulina spp., on the other hand, grew well at higher temperatures but showed the highest organic carbon production, and release to the medium, under low light conditions. These results led to a proposed process for the integration of algal ponding into an integrated system for the treatment of AMD. The algal biomass may be fed into the anaerobic digester as a carbon source, or it may be passed into a High Rate Algal Pond (HRAP) where it is stressed to enhance the organic carbon content. This can then be fed into the anaerobic digester as a carbon source. The impact of high levels of sulphide in the water feeding to the algal growth compartment was investigated. Spirulina spp. isolated from a tannery waste stabilisation pond was shown to be a sulphidophilic strain of cyanobacterium, capable of being adapted to high concentrations of sulphide. Dunaliella salina on the other hand was less tolerant. These results demonstrated the practical use of algal biomass providing an oxygen-rich cap for odour control on the surface of the facultative pond as well for the secondary treatment of sulphide-rich overflow to the High Rate Algal Pond. The ability of micro-algae to elevate the pH of their surrounding environment was evaluated as a functional precipitant and neutralisation reagent for acidic metal containing wastewater. Spirulina spp. was shown to perform effectively. D. salina was less functional in this environment. Anacystis spp. was effective in elevating the pH of a defined medium as well as a zinc-rich effluent. These results indicated the practicality of a neutralising function for algal ponds in the treatment of AMD. Metal removal in the system was found to be a combined function of sulphide precipitation, removal by binding to micro-algal biomass and extracellular polymeric substances. The feasibility of waste stabilisation ponding technology use for the treatment of large volume AMD effluents was provisionally demonstrated. It was shown that complex carbon sources would be used as efficient electron donors for sulphate reduction. The integration of algal ponding into the system provides for the generation of a sustainable carbon source, odour control with the recycling of oxygen-rich water onto the top of the facultative pond, secondary treatment of the anaerobic digester overflow, and the neutralisation of the incoming acidic effluents and removal of heavy metals. Integration of the individual unit operations, the feasibility of which has been provisionally demonstrated in this study, into a continuous process train is being investigated in follow-upstudies.
- Full Text:
- Date Issued: 1999
Evaluation of a 'defouling on demand' strategy for the ultrafiltration of brown water using activatable enzymes
- Authors: Buchanan, K
- Date: 1999
- Subjects: Water -- Purification , Ultrafiltration , Enzymes , Membranes (Technology)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3904 , http://hdl.handle.net/10962/d1003963 , Water -- Purification , Ultrafiltration , Enzymes , Membranes (Technology)
- Description: New approaches to the application of membranes for the production of potable water are constantly being sought after in anticipation of future demands for increasingly rigorous water quality standards and reduced environmental impact. A major limitation, however, is membrane fouling, which manifests itself as a continual reduction in flux over time and thus restricts the practical implementation to restore flux. Mechanical and chemical methods have been implemented to restore flux to ultrafiltration systems, but these either result in a break in the process operation or lead to membrane damage or additional pollution problems. This project was aimed to develop a 'defouling on demand' stategy for cleaning membranes used during brown water ultrafiltration. The process involves the use of activatable peroxidase enzymes, which were immobilised onto flat sheet polysulphone membranes. Following flux decline which reaches a critical level with the build-up of the foulant layer, the immobilised enzyme layer was activated by the addition of a chemical activator solution, in this case hydrogen peroxidase and manganous sulphate. Manganese peroxidase was found to be the most effective enzyme at alleviating fouling by degrading the foulant layer formed on the membrane surface and hence restored flux to the ultrafiltration system. A 93% flux improvement was observed when manganese peroxidase was activated when 800uM manganous sulphate, 100mM hydrogen peroxide were added in the presence of a manganese chelator, lactate. The concept and the potential benefits this system holds will be discussed in further detail.
- Full Text:
- Date Issued: 1999
Development and characterisation of a membrane gradostat bioreactor for the bioremediation of aromatic pollutants using white rot fungi
- Authors: Leukes, Winston D
- Date: 1999
- Subjects: Aromatic compounds Pollutants Fungi Bioremediation Industrial microbiology Biotechnology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4032 , http://hdl.handle.net/10962/d1004092
- Description: Bioremediation of aromatic pollutants using the ligninolytic enzymes of the white rot fungi has been thoroughly researched and has been shown to have considerable potential for industrial application. However, little success in scale-up and industrialisation of this technology has been attained due to problems associated with the continuous production of the pollutant-degrading enzymes using conventional bioreactor systems. The low productivities reported result from the incompatibility of conventional submerged culture reactor techniques with the physiological requirements of these fungi which have evolved on a solid-air interface, viz. wood. The enzymes are also produced only during the stationary phase of growth and can therefore be regarded as secondary metabolites. This study reports the conceptualisation, characterisation and evaluation of a novel bioreactor system as a solution to the continuous production of idiophasic pollutant degrading enzymes by the white rot fungus Phanerochaete chlysosporium. The reactor concept evolved from observation of these fungi in their native state, i. e. the metabolism of lignocellulosic material and involves the immobilisation of the organism onto a capillary ultrafiltration membrane. Nutrient gradients established across the biofilm, an inherent characteristic of fixed bed perfusion reactors, are exploited to provide both nutrient rich and nutrient poor zones across the biofilm. This allows growth or primary metabolism in the nutrient rich zone, pushing older biomass into the nutrient poor zone where secondary metabolism is induced by nutrient starvation. In effect, this represents a transformation of the events of a batch culture from a temporal to a spatial domain, allowing continuous production of secondary metabolites over time. Direct contact of the outer part of the biofilm with an air stream simulated the solid-air interface of the native state of the fungus. In order to facilitate the practical application of the membrane gradostat reactor (MGR) concept, conventional capillary membranes and membrane bioreactor modules were first evaluated. These were found to be unsuitable for application of the MGR concept. However, critical analysis of the shortcomings of the conventional systems resulted in the formulation of a set of design criteria for the development of a suitable membrane and module. These design criteria were satisfied by the development of a novel capillary membrane for membrane bioreactors, as well as a transverse flow membrane module, which is a novel approach in membrane bioreactor configuration. For the physiological characterisation of the MGR concept, a single fibre bioreactor unit was designed, which allowed destructive sampling of the biofilm for analysis. Using this system, it was shown that distinct morphological zones could be observed radially across the mature biofilm obtained through MGR operation. That these morphotypes do represent the temporal events of a typical batch culture in a spatial domain was confirmed by following the morphological changes occurring during batch culture of the immobilised fungus where the onset of primary and secondary metabolic conditions were manipulated through control of the nutrient supply. The different morphotypes were correlated to distinct growth phases by comparison of the morphology to the secretion of known enzymatic markers for secondary metabolism, viz. succinate dehydrogenase and cytochrome C oxidoreductase. Detailed structure-function analysis of the biofilm using transmission electron microscopy and adapted enzyme cytochemical staining techniques showed that the biofilm appeared to operate as a co-ordinated unit, with primary and secondary metabolism apparently linked in one thallus through nutrient translocation. This study provided new insights into the physiology of P. chrysosp,o rium and a detailed descriptive model was formulated which correlates well to existing models of wood degradation by the white rot fungi (WRF). Evaluation of the process on a laboratory scale using a novel transverse flow membrane bioreactor showed that a volumetric productivity of 1916 U.L.⁻¹day⁻¹ for manganese peroxidase, one of the pollutant degrading enzymes, could be attained, corresponding to a final concentration of 2 361 U.L.⁻¹ This may be compared to the best reported system (Moreira el at. 1997), where a volumetric productivity of 202 U.L.⁻¹day⁻¹was achieved with a final concentration of 250 U.L.⁻¹ However, MGR productivity is yet to be subjected to rigorous optimisation studies. The process could be operated continuously for 60 days. However, peak productivity could not be maintained for long periods. This was found to be due to physical phenomena relating to the fluid dynamics of the system which caused fluid flow maldistribution, which would have to be resolved through engineering analysis. In evaluation of the MGR concept for aromatic pollutant removal, in this case ρ- cresol, from growth medium, good performance was also achieved. The VmaxKm calculated by linear regression for the MGR was 0.8 (R² = 0.93), which compared favourably to that reported by Lewandowski et al. (1990), who obtained a Vmax/Km of 0.34 for a packed bed reactor treating chlorophenol. It was concluded that the MGR showed suitable potential to warrant further development, and that the descriptive characterisation of the biofilm physiology provided a sufficient basis for process analysis once engineering aspects ofthe system could be resolved.
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- Date Issued: 1999
Sulphate reduction utilizing hydrolysis of complex carbon sources
- Authors: Molipane, Ntaoleng Patricia
- Date: 1999
- Subjects: Sewage sludge , Acid mine drainage , Hydrolysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4000 , http://hdl.handle.net/10962/d1004060 , Sewage sludge , Acid mine drainage , Hydrolysis
- Description: Due to environmental pollution caused by acid mine drainage (AMD), the Department of Water Affairs has developed a National Water Bill for managing and controlling the water environment to prevent AMD pollution. The application of sulphate reducing bacteria have been demonstrated for the treatment of AMD. However, the scale-up application of this technology ultimately depends on the cost and availability of a carbon source. This study evaluated the use of sewage sludge to provide a carbon source for sulphate reduction in synthetic drainage wastewaters. The demonstration of this process in a laboratory-scale reactor proved that sewage sludge could provide a useful model and viable carbon source for evaluation of sulphate reduction as a process for treating AMD. Since sewage sludge is a complex carbon source, hydrolysis reactions controlling the anaerobic digestion of particulate substrate from this medium were optimized by evaluating the effect of pH on hydrolysis. Controlled and uncontrolled pH studies were conducted using a three stage mixed anaerobic reactor. Analysis of the degradation behaviour of the three important organic classes (carbohydrate, proteins and lipids) revealed that each class followed an indvidual trend with respect to pH changes. In addition, the solubilization of organic particulate carbon was also shown to be a function of pH. The hydrolysis pattern of organic substrate and COD solublization was induced at pH 6.5 rather than at high pH values (7.5 and 8.5). The biodegradation activity of sewage sludge was characterized by the API-ZYM1N test system to provide rapid semiquantitative information on the activity of hydrolytic enzymes associated with the degradation of carbohydrates, lipids, proteins and nucleic acids. A wide range of enzyme activities with phosphatases, aminopeptidases, and glucosyl hydralases dominating were displayed. The pattern of substrate hydrolysis correlated to the degradation efficiency of each organic class as a function of pH. The evaluation of scale-up application for sulphate reduction utilizing sewage sludge as a carbon source demonstrated that large water volume flows could possibly be treated with this cost-effective technology. Generation of alkalinity and sulphide in this medium was shown to be successful in the removal of heavy metals by precipitation. The use of this technology coupled to reduced cost involved showed that biological sulphate reduction utilizing hydrolysates of complex organic particulate from sewage sludge ss a carbon source has a potential scale-up application for the treatment of AMD.
- Full Text:
- Date Issued: 1999
The isolation and characterisation of thermostable hydantoinases from hydantoinase-producing bacteria
- Authors: Phehane, Vuyisile Ntosi
- Date: 1999
- Subjects: Hydantoin , Bacteria -- Physiology , Enzymes
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3998 , http://hdl.handle.net/10962/d1004058 , Hydantoin , Bacteria -- Physiology , Enzymes
- Description: In order to characterise thermostable hydantoin-hydrolysing enzymes from bacteria, locally-isolated thermophilic organisms were screened for the ability to convert hydantoin to N-carbamylglycine at 55°C using the hydantoinase enzyme. Cell disruption of a selected strain, RU-20-15, was conducted by French pressing to release enzyme from within the cell. In all of the experiments conducted, the amounts of product were low. In view of the low yields of products formed by the thermophiles, a previously-isolated Gram negative strain, RU-KM3L was selected from a number of mesophiles by screening for hydantoinase and carbamylase activity over a 40-55°C temperature range. Hydantoin conversion at 40°C using crude extract from pressed cells of this organism was similar to conversion at 50°C, and therefore subsequent assays were conducted at the higher temperature. The growth kinetics of RU-KM3L cells were studied and the enzyme activities of the extracts were compared in complete and chemically-defined media. The results suggested that the optimal time to harvest cells was at early stationary phase, when using complete medium for culture of cells; the specific activity of enzyme extracts produced by culture in complete medium was higher than that obtained in chemically-defined medium. 5-methylhydantoin was shown to be the preferred substrate for both the hydantoinase and carbamylase enzymes in the crude extract of RU-KM3L. The substrate specificity of the hydantoinase and carbamylase enzymes of the crude RU-KM3L extract was observed to be altered in the presence of increasing amounts of hydantoin, 5,5-dihydrouracil (DHU) and 5-thiouracil (TU) as inducers, showing selectivity for 5-methylhydantoin over hydantoin at inducer concentrations of 0.1 to 1%. A limiting effect on the hydrolysis of 5-methylhydantoin was observed when DHU and 5,5-dimethylhydantoin (DMH) were used as inducers, while the limiting effect on hydantoin specificity was observed when DHU and TU were used as inducers. The limiting effect was observed to be dependent upon the concentration of inducer, and was not observed when hydantoin was used as an inducer. The optimal time for assay of the hydantoinase enzyme in crude extract preparations at 50°C was observed to be 3h. Alkaline conditions were shown to be optimal for both the hydantoinase and carbamylase enzymes of RU-KM3L. Assay for enzyme activities of RU-KM3L extract in the presence of metal ions showed Mn²⁺ ions (and to a lesser extent, Co²⁺) to activate both the hydantoinase and carbamylase activities. Cu²⁺ ions were observed to inhibit the hydantoinase enzyme. In order to determine the location of the enzymes within the cell, cell debris from disrupted cells of RU-KM3L was removed by centrifugation. A decrease in enzyme activity in the supernatant was observed, and suggested association of the enzymes with the cell membrane. Ammonium sulfate fractionation experiments conducted on the crude extract provided further evidence for this result. Sonication of the crude enzyme extract was the only successful method for the releasing of membrane-associated enzyme. Of a number of strategies investigated, the use of sucrose at 50% (w/v) concentration was shown to preserve the hydantoinase and carbamylase enzyme activities during lyophilisation. Furthermore, assay for these enzyme activities showed the activities to be higher after lyophilisation in the presence of sucrose. However, sucrose did not increase the thermostability of lyophilised crude enzyme extracts. Water-miscible organic solvents at 1% concentration were shown to be inhibitory to the hydantoinase and carbamylase enzymes of RU-KM3L, and the inhibition was also observed to increase with increasing concentrations of these solvents. Hydantoinase activity in the presence of water-immiscible organic solvents was shown to increase with an increase in the hydrophobicity of these solvents, but the activity observed was not significantly higher than activity in the absence of solvent when hydantoin and 5-methylhydantoin were used as substrates. The possibility of reversing the hydantoinase enzyme reaction by water-immiscible organic solvents was investigated, and the results obtained suggested that the reaction could be reversed. It was thought that the partitioning of substrates or products into hydrophobic organic solvents could influence the reaction equilibrium, but the partitioning observed was not sufficient to affect reaction rates. Peptide synthesis was shown to have occurred in small amounts when the hydantoinase reaction was carried out in the presence of water-immiscible organic solvents. In conclusion, the hydantoin-hydrolyzing enzyme activity of a crude extract preparation from the bacterial strain RU-KM3L was characterised at elevated temperatures, and in the presence of watermiscible and -immiscible organic solvents.
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- Date Issued: 1999
Removal of lead from solution by the non-viable biomass of the water fern Azolla filiculoides
- Authors: Sanyahumbi, Douglas
- Date: 1999
- Subjects: Azolla , Heavy metals -- Absorption and adsorption , Lead , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3901 , http://hdl.handle.net/10962/d1003960 , Azolla , Heavy metals -- Absorption and adsorption , Lead , Water -- Purification -- Biological treatment
- Description: The removal of lead from aqueous solution and lead-acid battery manufacturing waste-water by the non-viable biomass of the water fern Azolla filiculoides was investigated in both batch and column reactors. The maximum lead uptake by the Azolla biomass at a pH value of approximately 5, was found to be 100 mg lead/g biomass from aqueous solution. Lead removal varied from 30% of the initial lead concentration at pH 1.5 to approximately 95% at pH values of 3.5 and 5.6. Lead removal from aqueous solution decreased to 30% of the initial lead concentration if the lead concentration was initially over 400 mg/l. At initial lead concentrations of less than 400 mg/l, percentage lead removal was found to be over 90% of the initial lead concentration. Lead removal remained at approximately 90% between 10°C and 50°C. Biomass concentration (4-8 mg/l) had little effect on lead removal. The presence of iron (Fe) and lead, copper (Cu) and lead or all three metal ions in solution at varying ratios to each other did not appear to have any significant effect on lead removal. Percentage lead, copper and iron removal from aqueous solution was 80-95, 45-50 and 65-75% respectively for the different multiple-metal solutions studied. No break-through points were observed for lead removal from aqueous solutions in column reactors, with initial lead concentrations of less than 100 mg/l at varying flow rates of 2, 5 and 10 ml/min. This suggested that flow rate, and therefore retention time, had little effect on percentage lead removal from aqueous solution, which was more that 95%, at low initial lead concentrations (less than 100 mg/l). At initial lead concentrations of 200 mg/l or more, an increase in flow rate, which equates to a decrease in column retention time, resulted in break-through points occurring earlier in the column run. Percentage lead removal values, from lead-acid battery efiluent in column systems, of over 95% were achieved. Desorption of approximately 30% and 40% of bound lead was achieved, with 0.5 M HNO₃ in a volume of 50 ml, from two lead-acid battery. Repeated adsorption and desorption of lead by the Azalia biomass over 10 cycles did not result in any decrease in the percentage lead removal from effluent, which strongly suggested that the Azalla biomass could be re-used a number of times without deterioration in its physical integrity, or lead removal capacity. No evidence of deterioration in the Azolla biomass's physical integrity after 10 successive adsorption and desorption procedures was observed using scanning electron microscopy. The Azolla filiculoides biomass was, therefore, found to be able to effectively remove lead from aqueous solution and lead-acid battery effluent repeatedly, with no observed reduction in it's uptake capacity or physical integrity.
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- Date Issued: 1999
The removal of toxic heavy metals from aqueous solutions by algal extracellular polysaccharides
- Authors: Selepe, Mamaropeng Marcus
- Date: 1999
- Subjects: Heavy metals -- Absorption and adsorption , Copper , Lead , Algae -- Biotechnology , Polysaccharides -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3994 , http://hdl.handle.net/10962/d1004054 , Heavy metals -- Absorption and adsorption , Copper , Lead , Algae -- Biotechnology , Polysaccharides -- Biotechnology
- Description: This study investigated the possible use of algal extracellular polysaccharide as a biosorbent for removal of heavy metals (copper and lead) from aqueous solutions as a means of bioremediation for metal containing effluents. This biopolymer has good biosorbent properties and a potential to provide a cost effective, selective and efficient purification system. A variety of environmental conditions induce the production of extracellular polysaccharides in algae. The production of exopolysaccharides by Dunaliella cultures was induced by nitrogen deficient conditions. A high ratio of carbon to nitrogen source considerably enhanced the polysaccharide release. Purified extracellular polysaccharide samples exhibited a monosaccharide composition consisting of the following sugars: xylose, arabinose, 2-0-methyl mannose, mannose, glucose and galactose. The relative abundance (%) of these sugars were calculated relative to xylose. The major sugar constituent was 2-0-methyl mannose, which was present at approximately 160% relative to xylose. The percentage relative abundance of other sugars was as follows: 18.8; 86.8; 85.3 and 22.3% for arabinose; mannose; glucose and galactose respectively. The identity of the various constituents were confirmed by mass spectrometry. The ability of Dunaliella exopolysaccharides to accumulate metals was investigated. The following parameters were studied because they affect metal uptake: solution pH, biomass concentration, temperature, time and metal concentration. The uptake of both copper and lead were pH dependent. However, metal uptake was not significantly affected by temperature. Kinetic studies showed that Dunaliella extracellular polysaccharides exhibit good bioremediation properties. Metal uptake was rapid. In addition, the exopolysaccharide has good metal binding capacity with an uptake capacity for lead of 80 mg/g from a solution containing initial lead concentration of approximately 40 mg/l. Competition studies revealed that the presence of a second metal in solution inhibits uptake of the other metal compared to uptake in single metal solution of that particular metal. The presence of lead inhibited the uptake of copper from approximately 65% in single metal solution to 10% in binary metal solution. The presence of copper also inhibited lead uptake, though not to the same extent. Higher concentrations of lead could not completely prevent removal of copper from solution and visa versa. The same was true for lead which could not be displaced by a four-fold concentration of copper. Instead, a certain percentage of copper was always removed showing that lead did not compete with copper for these binding sites. In conclusion it appears that, copper and lead bind to different sites on Dunaliella exopolysaccharides and that they exhibit selective or preferential removal of lead.
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- Date Issued: 1999
An investigation into the neuroprotective properties of melatonin
- Authors: Southgate, Garrick Steven
- Date: 1999
- Subjects: Melatonin
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3900 , http://hdl.handle.net/10962/d1003959
- Description: Until the beginning of this decade the neurohormone, melatonin, had been considered as little more than a tranquillising hormone, responsible for regulating certain circadian and circannual rhythms. In the last eight years, a whole new dimension to melatonin’s role in biological organisms has emerged. In 1991 it was discovered [1,2] that melatonin exhibited antioxidant properties. Since then, many researchers [3,4] have found melatonin to be a powerful free radical scavenger and antioxidant. In the present study, the ability of melatonin to offer neuroprotection against glutamate, N-methyl-D-aspartate (NMDA), quinolinic acid (QA) and kainic acid (KA) (collectively referred to as the glutamate receptor agonists) was investigated. It was first shown that stress causes an increase in circulating glucocorticoid concentrations, which resulted in an increase the number of glutamate receptors on synaptic membranes in rat brain homogenate. Melatonin acted to reduce the number of glutamate receptors present on the synaptic membranes, implying that melatonin has neuroprotective properties, as overstimulation of the glutamate receptors leads to excitotoxicity and neurodegeneration. Further investigations showed that the glutamate receptor agonists induce neurodegeneration in primary neuronal cell cultures. Both co-treatment and posttreatment with melatonin against the glutamate receptor agonists, increased neuronal cell viability in a dose dependent manner. Melatonin also appeared to offer protection against quinolinic acid-induced neurodegeneration following intrahippocampal injections of quinolinic acid. The mechanism whereby melatonin offered this protection was investigated. The glutamate receptor agonists caused an increase in intracellular calcium concentrations, which is known [5] to be responsible for initiating the excitotoxic response. Melatonin had no effect on regulating intracellular calcium concentrations Additional studies indicated that melatonin was effective at scavenging superoxide radicals. Production of superoxide radicals was induced by the glutamate receptor agonists in primary neuronal cultures. Superoxide radicals induce lipid peroxidation, which involves the destruction of lipid membranes by chain reactions. By acting as an antioxidant, melatonin was able to reduce quinolinic acid-induced lipid peroxidation in rat brain homogenate, in a dose dependent manner. Melatonin was also effective at reducing lipid peroxidation induced by the glutamate receptor agonists in primary neuronal cultures. Melatonin therefore appeared to be offering neuroprotection by removing superoxide radicals and inhibiting lipid peroxidation. It had been reported [6] that melatonin inhibits nitric oxide synthase activity. This enzyme produces the free radical, nitric oxide, and can also produce superoxide radicals. Melatonin was able to reduce nitric oxide synthase activity in a dose dependent manner. This is a novel method of neuroprotection, as melatonin was now acting as an enzyme regulator. The results obtained demonstrate that melatonin offers neuroprotection against glutamate induced excitotoxicity, by removing free radicals and preventing lipid peroxidation. The neurohormone offers further protection by decreasing the activity of enzymes that aid in the neurotoxic cascade. Melatonin is the most potent naturally occurring free radical scavenger in the body [3]. During aging, the serum concentrations of melatonin decrease [7]. During the senescence of life, free radical damage to the body is at its highest [8], while at the same time melatonin concentrations are at their lowest. Melatonin therefore shows potential for the treatment of diseases and disorders that exhibit an excitotoxic pathology.
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- Date Issued: 1999
The role of vitamin E succinate in regulation of growth and cyclooxygenase expression in B16 murine melanoma cells
- Authors: Van der Merwe, Adele Shanette
- Date: 1999
- Subjects: Melanoma , Vitamin E
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3988 , http://hdl.handle.net/10962/d1004047 , Melanoma , Vitamin E
- Description: This study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate supplementation on B16 murine melanoma cell growth in vitro. Studies revealed that supplementation of 5, 7 and 10µg/ml of this vitamin significantly inhibited growth of B16 cells. Non-malignant LLCMK cells supplemented with the same concentrations of vitamin E succinate resulted in similar inhibition of cell growth. The actual mechanism by which vitamin E succinate inhibits B16 cell growth is unclear, though there has been much speculation about its possible role as an antioxidant. Vitamin E succinate is not a physiological antioxidant and for this ester to behave as an antioxidant, cleavage of the ester bond must occur, releasing the antioxidant vitamin E part of the molecule. To determine whether the observed inhibitory effects on B16 cell growth were due to the intact vitamin E succinate or the vitamin E cleavage product, cleavage studies were undertaken. Results from these studies revealed that in B16 cells vitamin E succinate cleavage did not occur suggesting that the observed inhibitory effects of vitamin E succinate on B16 cells were due to the intact compound. In contrast vitamin E succinate cleavage was shown to occur in LLCMK cells, suggesting that these cells may contain an esterase capable of liberating succinic acid and vitamin E. Further studies focussed on the possible role of vitamin E succinate in regulation of cyclooxygenase activity in B16 cells as vitamin E succinate was found to effect the activity of various enzymes involved in the arachidonic acid cascade, notably cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis. Time course studies were used to determine when the cyclooxygenase protein was being produced, thus allowing an estimation of when the gene was being 'switched on'. These studies revealed that vitamin E succinate does not significantly effect cyclooxygenase activity in B16 cells over a period of 2 to 12 hours as compared to the OE control cultures. Further studies using RNA techniques investigated whether vitamin E succinate was having an effect on cyclooxygenase activity at a molecular level. These investigations were unsuccessful for the 6 day supplementation for a number of possible reasons, the main reason being RNA stability. Subsequent studies revealed an increase in COX mRNA after 2 hours, suggesting that the gene was 'switched on' soon after supplementation with vitamin E succinate, and further increases in COX mRNA were observed after 8 to 12 hours. The molecular studies were, however, inconclusive. Previous studies suggested that vitamin E succinate was indirectly causing growth inhibition of B16 cells via regulation of cyclooxygenase activity, however, this study does not support these findings and it would seem unlikely that regulation of cyclooxygenase expression in B16 cells by vitamin E succinate has a role to play in the mechanism by which vitamin E succinate inhibits growth in B16 cells.
- Full Text:
- Date Issued: 1999
The effect of combined vitamin E succinate and ascorbic acid supplementation on growth and cyclooxygenase expression in murine melanoma (BL6) cells
- Authors: Van Rooyen, Megan Lynne
- Date: 1999
- Subjects: Vitamin E , Vitamin C , Melanoma
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3982 , http://hdl.handle.net/10962/d1004041 , Vitamin E , Vitamin C , Melanoma
- Description: This thesis examines the effect of combined vitamin E succinate and Asc supplementation on the in vitro growth of a non-malignant monkey kidney (LLCMK) and a malignant melanoma (BL6) cell line, with nutritional concentration ranges of 5-20µg/ml and 25-50µg/ml respectively. Vitamin E and C are thought to interact synergistically to inhibit tumour cell growth by virtue of their antioxidant properties, whereby they quench free radicals and terminate lipid peroxidation. Furthermore vitamin E and C are thought to modulate the biosynthetic pathways in arachidonic acid metabolism at a number of different points. This may also offer a means of regulating tumour cell growth. It is well documented that vitamin E and C are distributed in the lipid and aqueous phases in the cell respectively. However, the cells need to obtain the vitamins from the environment in which they are found in order to exert a growth inhibitory effect. Supplementation of combined vitamin E succinate and Asc on BL6 and LLCMK cells resulted in a significant increase in LLCMK cell growth, and a significant decrease in cell growth was observed in BL6 cells. Vitamin E succinate in its esterified form cannot function as an antioxidant and requires the cleavage of the succinate to become an active antioxidant. The metabolism of vitamin E succinate to form free vitamin E in LLCMK and BL6 cells resulted in the cleavage of the succinate group from the vitamin E molecule in BL6 cells only, thus suggesting that an esterase may be present in BL6 cells. This would allow for a synergistic interaction between the two vitamins. The arachidonic acid cascade generates a family of bioactive lipids that modulate diverse physiological and pathological responses including tumour growth and promotion. The enzyme prostaglandin endoperoxide synthase (PGHS) or cyclooxygenase (Cox) is the key enzyme in the biosynthetic pathway leading to the formation of prostaglandins. Two enzyme isoforms of Cox have been identified, Cox 1 and Cox 2. Supplementation with vitamin E succinate and Asc at a combination 20:25µg/ml respectively resulted in a trend of increasing Cox activity over 12 hours suggesting that vitamin E and Asc have a stimulatory effect on Cox activity in BL6 cells. The inhibitors of Cox 2, dexamethasone, showed a decreasing trend in Cox activity at the 20:25µg/ml combination, while cycloheximide showed an initial stimulatory effect and then a gradual decrease in Cox activity. The elimination of the Cox activity by dexamethasone suggests that transcriptional regulation may be occurring in BL6 cells. We examined by Northern blot analysis whether combined supplementation of vitamin E succinate and Asc caused an elevation of Cox 2 RNA expression in BL6 cells. An inducible effect of Cox 2 was observed after 2 hours of supplementation with a combination of vitamin E succinate and Asc in BL6 cells, however the results are inconclusive and further studies are required to substantiate this finding.
- Full Text:
- Date Issued: 1999